DE10032568A1 - Device and method for electrically contacting biological cells suspended in a liquid - Google Patents

Abstract

The invention relates to a device for making electrical contact with biological cells (11), suspended in a fluid (14), comprising a substrate with at least one opening and a means of immobilising a cell (11) on the opening. Furthermore, an electrode for making electrical contact with the immobilised cell (11) is provided. A contact unit with a contact point (23) is arranged at the opening, the upper end of which extends into the opening, such that the said end contacts the cell membrane of an immobilised cell (11). The contact point (23) comprises a contact channel opening at the upper end thereof, by means of which a hydrodynamic vacuum may be played on the cell membrane and the electrode is in electrical contact with the contact channel. The invention further relates to a method for making electrical contact with biological cells suspended in a fluid, whereby a cell is immobilised above an opening in a substrate and the cell contacted by at least one electrode. In order to make contact with the immobilised cell (11), a contact point (23), extending through the opening, produces a hydrodynamic vacuum on the cell membrane of the cell (11), whereby the electrode makes electrical contact with the cell (11) by means of the contact point (23).

Description

The present invention relates to a device for elec trical contact in suspension in a liquid located biological cells, with at least one opening tion having substrate, with means for immobilizing egg ner cell on the opening, preferably one in the opening protruding intake duct, through which a hydrodynami at the opening negative pressure to immobilize a cell on the opening can be generated, and at least one electrode for the electri Contacting the immobilized cell.  

The invention further relates to a method for electrically contacting a biological cell in suspension in a liquid, comprising the steps:

• - Immobilize a cell above one in one Substrate provided opening, preferably by creating a hydrodynamic vacuum, and
• - Contacting the immobilized cell via at least one Electrode.

An apparatus and a method of the above Art are known from DE 197 12 309 A1.

The known device comprises a microelectrode arrangement, where the cells are captured in microcuvettes, de There is an electrode on the bottom. The electrode is with a central intake duct, in which suitable Connecting channels that run below the electrodes Vacuum can be generated. In this way it is possible pull individual cells towards the electrodes and they close to the electrodes under a certain contact pressure immobilize. With the known device Mes solutions on the cells, but only from the outside of the membrane forth.

Such microelectrode arrangements are generally the Un tests of biological cells used. The microelectro the arrangements serve z. B. to stimulate the cells or to derive potentials. The investigations can be done in in a biological environment or in an artificial environment  be performed. The arrangements include in egg Nem carrier body, ie on a substrate, a variety of Microelectrodes, the dimensions of which are of the order of magnitude of cells are in the range of a few µm to a few 10 µm.

In conventional microelectrode arrangements, one is more or less dependent on chance, whether one or the other another cell on a particular electrode or Not. In practice, the cells can generally only be partially covered on an electrode so that the stimulation of the cell or the derivation of a cell potenti than is limited to this partial area.

These disadvantages are caused by those mentioned in the introduction DE 197 12 309 A1 known device for the most part tigt, in particular it is ensured that on a microelectr trode only one cell comes to rest. On the other hand, it remains still left to chance whether a cell and which Cell is captured in the microcuvette. It is also not to be ruled out that parts in the liquid be sucked into the microcuvette, although the through enable a measurement to be carried out, but the results are not usable.

Another disadvantage of the known device and the be known method is that only measurements on the Au can be carried out on the outside of the cell membrane. Intrazel lular measurements are not possible.  

Such intracellular measurements can, however, be made with the perform so-called patch clamp technology, in which a so-called called membrane spot clamp is used; see e.g. B. Neher and Sakmann: "The study of cell signals with the patch Clamp technology ", Spectrum of Sciences, May 1992, p. 48.

This technique uses an electrically conductive liquid filled micropipette targeted to an adherent cell cited and placed on their membrane, so that this bulges slightly inwards. Thereupon the of the pipettes tip trapped membrane stain slightly due to negative pressure sucked, causing this to the surrounding liquid sealed and electrically isolated. This isolation will also known as "Gigaseal".

In this way it is possible e.g. B. Measurements on ion channels to be carried out on the outside of the membrane.

However, if the sealed membrane continues to be sucked in, the membrane spot is perforated, so that a hydrodynamic and electrically sealed from the surrounding liquid There is access through the pipette opening to the cell interior, so that intracellular measurements and stimulations are possible (so-called "whole cell patch").

The disadvantage of the patch clamp technique is that it only works adherent or otherwise immobilized cells performed to which the fragile glass pipette can be attached using an egg Nes micromanipulator must be introduced. For this reason the number of cells that can be contacted simultaneously is extremely limited,  so that several cells are only processed sequentially can be.

This method is therefore unsuitable for mass examinations, as they e.g. B. in the field of pharmaceutical screening, substance screening ning etc. would be required.

Another disadvantage with conventional patch clamp Technology is that it can not be automated, son which is carried out manually by personnel, the great experience bring along and have a lot of tact.

From DE 198 27 957 A1 it is known to have one cell on one Immobilize glass supports, the surface of which is a ring structure has door-shaped profiling in order to ver the cell adhesion improve. On the glass support is in the area of the support surface a cone-shaped projecting electrode is arranged for the cell net with its sharp, ring-shaped tip into the cell cuts the membrane and pre-cuts for intracellular measurements hen is. The electrode can be used as a cylindrical electrode with a Hollow channel can be designed, through which an Un on the cell membrane terdruck to fix the cell and / or perforate the cell membrane can be exercised.

How the cell gets on the support area is not wrote. A disadvantage of the known device is that the electrical seal on the ring-shaped electrode tip ze only by the "holding force" of the cell on the profiled Surface is determined. Carrying out mass investigations chungen is not possible with the known device.  

Against this background, the present invention Task based on a method and an apparatus of the gangs mentioned in such a way that the first disadvantages mentioned can be avoided. In particular, should an automatable, efficient and universally applicable Possibility for electrical contacting of the cells will be able to make measurements both on the outside of the To be able to perform membrane as well as intracellularly. The new The process should be simple and automatable be, the new device is structurally simple should be.

In the device mentioned at the beginning, this task is there solved by that at the opening a contact unit with a Contact tip is arranged, the upper end so in the public protrudes that it has a cell membrane of an immobili sied cell comes into contact, the contact tip one at ih Rem has the upper end opening contact channel, on the the cell membrane can be subjected to a hydrodynamic negative pressure, and the electrode is electrically connected to the contact channel stands.

In the method mentioned at the beginning, this makes this task solved that by contacting the immobilized cell a contact tip protruding into the opening onto the Cell membrane of the cell exerted a hydrodynamic negative pressure with the electrode through the contact tip the cell comes into electrical contact.

The object of the invention is based on this Wei se completely solved.  

Regardless of the immobilization, preferably by Un ter pressure in the intake duct, protrudes into the opening Patch clamp tip in, over the outside of the membrane and / or electrical signals measured intracellularly and / or can be supplied. The holding pressure is independent and separate from the contact pressure so that the contact tip is egg NEN can have a very small diameter that a verver casual gigaseal and good perforation. The Diameter of the opening is significantly larger, so that a very large holding pressure results without the cell in the An suction channel or even drawn into the contact channel.

In this way, stable long-term measurements are also possible under Per fusion possible if the cell is washed with substances on the outside to induce certain cell responses that like which are to be detected via the electrode.

Because the contact tip protrudes into the opening, it results when the cell is sucked onto the opening by means of the suction ka nals an arching of the cell membrane in the area of contact great, as would also be the case with the manual patch clamp technique is worth it to ensure a good gigaseal, if a slight negative pressure on the membrane through the contact channel stain is exercised. With a short, intermittent increase of the negative pressure in the contact channel can be known Perforate the membrane stain reproducibly.

The inventors of the present application have recognized that by separating the intake duct and the contact duct casual and therefore automatable positioning of cells  over the opening and its contact after the patch clamp Technology becomes possible.

From the older, but not prepublished DE 199 48 473 A1 discloses a device in which one Cell through an intake port to an opening in one Substrate is sucked, with a downstream in the suction channel Electrode is arranged over the electrically conductive Liquid in the intake duct measurements both on the outside side of the cell membrane as well as intracellular measurements light, if by a short pressure surge that of the on suction channel enclosed membrane spot was perfused.

In this device there is only one channel, which both serves as a suction as well as a contact channel and therefore a re must have a relatively large inner diameter in order to correspond to be able to exert sufficient holding force on the cell. It will a Gigaseal here too by applying the negative pressure testifies, however, according to the inventors, this is not the case reliable as in the device according to the invention. Further there is no arching in the membrane of the suction Cell, rather the membrane is rather curved outwards and drawn into the intake duct, so that the effective membrane area can change, so that no stable Long-term measurements are possible.

In a development of the device according to the invention, it is preferred if the contact unit is removable at the opening is ordered, with the contact tip also preferably being made is interchangeably arranged.  

It is advantageous here that the contact unit and / or the con clock tip can be easily replaced while the practice Other components of the new device reusable are. This measure is based on the knowledge that patch Clamp tips can only be used once, already at a second use does not result in a sufficient Gigaseal or no reliable membrane perforation achieved.

It is preferred if the suction channel in the substrate runs, on the other hand, the intake duct at least from can run in sections in the contact unit.

If the suction channel runs in the substrate, it is advantageous that the contact unit is very simple and inexpensive can be. In contrast, the intake duct at least runs off in sections in the contact unit, so the external connection both the intake duct and the contact duct in a simple manner Way done on the contact unit itself, what the handling and simplified the structure.

Furthermore, it is preferred if the contact unit is replaced cash, the contact tip having top and a like the usable, the electrode and a first portion of the Has contact channel lower part, the Kon clock unit preferably in the area of the opening on the outside The substrate rests and the opening is liquid-tight to the outside completes, further preferably the upper part under pressure outside on the substrate and the lower part under pressure on the Top fits.  

These measures have the advantage that to replace the Contact tip only out the top of the contact unit must be changed. Because bottom part and top part together or are under pressure on the substrate are not screw work or similar manipulations required the out exchange can be automated e.g. B. with the help of a micromanipula tors easily done.

It is preferred if a sealant is provided between the substrate and the upper part and / or between the upper part and the lower part, which is preferably selected from the group: silicone paste, two-component elastomer and poly dimethylsiloxane elastomer (Sylgard ^TM ) ,

This measure has the advantage that even at low Print a good liquid tight seal between Substrate, upper part and lower part is made so that the micro manipulator both in terms of accuracy and respect pressure requirements are lower than if, for example, an O-ring was used for sealing.

It is further preferred if there is a chamber in the lower part seen into which the electrode protrudes and the first Ab cut of the contact channel opens.

The advantage here is that the electrode and the first section of the contact channel in the reusable lower part are net, so the cost of performing a measurement can only be determined by the replaceable upper part that le diglich the contact tip and a second section of the Contact channel carries.  

In this context, it is preferred if in the top one assigned to the chamber and passing through the contact tip funnel-shaped second section of the contact channel arranged is.

It is further preferred if between the upper part and the A ring is formed around the contact tip, which has an opening pointing towards the lower part, which a first section of the intake duct in the lower part is arranged.

In this simple way, the suction channel in the contact unit can be integrated. The intake duct now runs from that Lower part over the opening in the annulus around the contact point and suck the cell there on the opening and against the Contact tip. It is also advantageous here that only one very simply constructed top, which like all other compos components in microsystem technology, i.e. microstructured can be replaced.

In a further development, it is preferred if the annular space is open the substrate is sealed with a membrane, in of a passage surrounding the contact tip at a distance is seen.

The advantage here is that the opening in the substrate is clear can be larger than the passage through which the intake duct the cell sucks on the opening. In this way, the Manipulation accuracy of the micromanipulator lower requirements to make, since it only has to be ensured that the Passage is arranged in the region of the opening, the seal  to the outside through the membrane, which is advantageously on the top is attached.

In general, it is preferred if a Reference electrode protrudes into the liquid.

This measure has the advantage that the spatial Proximity of electrode and reference electrode very reliable and reproducible potentials on the cell membrane or intracellu can be measured.

In general, it is preferred if on the substrate around the opening a functional coating as a means of real estate bilization of the cells is provided.

This measure advantageously serves to select the cells to be contacted. The measure is at a generic device and a generic method also in itself new and inventive. By using a functional coating it is now possible from a sus Pension different cells to a certain type of cells select and contact. Only the cells that are on the the cells self-coordinated functional coating immo are also used for measurement.

The functional coating is selected from the Group: polyanions; polycations; polyethyleneimine; antibody against cell surface molecules such as integrins (e.g. fibronectin) or lectins; and magnetic coating.  

By choosing the polyanions, polycations and polyimines appropriate chemical immobilization is provided, while by immobilizing suitable antibodies in the functional coating a specificity for any Cell surface molecules can be made.

If the functional coating comprises a magnetic coating device, it is possible in this way to capture magnetic antibodies which have previously been added to the cell suspension and have accumulated on certain cell surface molecules. By choosing a special magnetic antibody, precisely predeterminable cells from the cell suspension can be immobilized in this way. Antibodies coupled to magnetic particles are known from the prior art; they are sold, for example, by Per Septive Diagnostics Incorporated, Cambrigde, Massachusetts; see Ahern: "Antibodies Making Their Way From The Clinic To The Research Lab", The Sci entist, Volume 9 , 1995 , pp. 18-19.

It is further preferred if a positioning unit is provided to position a cell over the opening ren, wherein the positioning unit comprise a laser tweezers can build up an electromagnetic field or a hydrody can generate namic force.

The positioning unit can therefore already on the one hand by the Intake duct can be represented by a hydrodynamic force generated, but also independently of the intake duct or in addition to the intake duct z. B. a laser tweezers or a electromagnetic field can be provided.  

Laser tweezers are generally known from the prior art knows, these optical tweezers are opti manipulators based on the conservation of momentum.

An object that is largely transparent to laser light barely bears energy, but directs the beam after refraction law from the original direction. This refraction over carries a pulse on a cell that moves the cell. On Pair of symmetrical laser beams thus forms a trap in which the cell is held. By moving the rays you can move the cell; see Berns: "With laser tools into the cell Inner ", Spectrum of Sciences, July 1998, p. 56.

By means of the positioning unit z. B. a cell on the functional coating can be put on. The cell will be there released by the positioning unit, it remains only over the opening when through the functional coating can be immobilized. In this way one can make simple selection of cells. If the cell from the Transport flow in the device after being released moving the positioning unit has a negative selection on, otherwise a positive selection.

In this way, a suspension can be differentiated cells select exactly the cells that are connected to the device tion should be contacted. This selection is also included a generic device and a generic The process itself is new and inventive.

It is preferred if the substrate is integral is part of a wall of a flow channel through which the  Liquid with defined and / or adjustable transport Flow is directed to the opening, preferably a Sensor unit is provided which is based on a parameter of the cell responds and the positioning unit and / or the intake duct controls.

The sensor unit also serves for the selection of cells and is taken individually as well as the functional coating a device and a method of the generic type new and inventive.

The parameter is selected from the group: optical Pa parameters such as fluorescence, absorption, reflection, Diffraction, refraction; geometric parameters such as Size, length, diameter, shape; electrical parameters as for for example capacity, resistance; and biological, chemical, physical cell parameters.

The sensor unit is preferably upstream of the Opening arranged.

The sensor unit enables in particular in connection with the adjustable transport flow and / or the position a selection of cells. If the sensor unit a cell to be contacted based on the measured cell parameters le recognizes this to the positioning unit and / or the Intake duct passed on, which is then set knowing the transport flow at the time when the cell selected by the sensor unit rich located. It is also possible just before the intake duct to provide a further sensor unit for localization  serves a cell and its current location on the intake duct and / or the positioning unit reports.

The sensor unit as well as the further one that may be provided Sensor unit thus enable a significantly more efficient Se lesson of cells as the functional coating, where by combining sensor unit (s), positioning unit and functional coating a very sharp selection of the contacting cells is possible.

Generally it is preferred if upstream of the opening a micro perfusion unit is arranged to immobilize Washed cell with test substances, preferably the components of the device in microsystem technology represents are.

In this way it is possible to use the immobilized cell to wash up small amounts of substance, so that cost-saving gear can be processed.

In the new method, it is preferred if before each con clock the contact tip is exchanged.

The advantage here is that for each new contact a new one Contact tip is used for safe training of a gigaseal as well as a reliable perforation of the um closed membrane stain, if desired.

It is further preferred if the liquid is adjustable Transport flow directed through a channel to the opening becomes.  

The advantage here is that the suspended cells in known Swim past the opening so that there is a drain control can actuate the intake duct in good time.

It is further preferred if the cell adheres to a cell functional coating is immobilized around the public voltage is arranged around on the substrate, the cell preferably by a positioning unit over the opening po is sitioned and further preferably before positioning a cell a parameter of the cell measured and dependent The positioning cell depends on the parameter is chosen.

The advantage here is that not with completely pure cell popula tion must be worked, but that also from mixed Cell suspensions selected desired cell types for contacting can be chosen.

It is preferred if a cell is selected as a result is that it uses the positioning unit on the functio positioned and then from the position kidney unit is released so that only cells are immobilized remain that respond to the functional coating.

The advantage here is that almost everyone on the positioning unit floating cell over the functional coating thereupon it is tested whether or not it should be contacted can be released.  

It is preferred if the cell is in addition to the radio tional coating by hydrodynamic vacuum immo is provided.

This measure has the advantage that a reliable holding force on a cell selected via the functional coating is selected so that during a long-term measurement z. B. can be held securely with perfusion.

Finally, it is still preferred if the cell from the public voltage is removed by the Un is built up to an overpressure.

The advantage here is that after the end of a measurement and stimula phase, the cell can be actively rejected. In summary, the new device and the new Ver So drive the possibility of single cells from a suspension according to at least one criterion, i.e. a cell parameter select and the selected single cells from the Suspensi to be positioned at a discharge point, i.e. at the opening and immobilize. The immobilization takes place either the one via an intake duct, i.e. with a hydrodynamic sub printing, and / or via a functional coating.

According to the procedure of the classic patch clamp technique becomes the cell for the purpose of forming a high-key seal sucked through the contact channel, the sucked in and down sealed membrane optionally broken by a pressure pulse can be.  

The immobilized and contacted cell can be filled with liquid can be perfused, which contain test substances, so that Serial examinations on many cells can be automated can be performed, while taking advantage of classic patch clamp technology can be used.

Further advantages result from the description and the at added drawing.

It is understood that the above and the following standing features to be explained not only in each specified combination, but also in other combinations or can be used alone, without the scope of to leave the present invention.

Embodiments of the invention are in the drawing are shown and are described in more detail in the following description explained. Show it:

Figure 1 is a schematic overview of the new Vorrich device.

FIG. 2 shows an enlarged illustration of a section of the device from FIG. 1 in the area of the contacting and measuring unit; FIG.

Figure 3 shows the schematic flow of an electrical contact processing a cell and the exchange of the contact tip.

Fig. 4 shows a first embodiment of a replaceable contact tip, wherein the suction duct is arranged in the substrate;

FIG. 5 shows an embodiment as in FIG. 4, but in which the contact unit is divided into an exchangeable upper part and a reusable lower part;

FIG. 6 shows a contact unit as in FIG. 5, but the suction channel is formed in the contact unit; and

Fig. 7 shows a contact unit as in Fig. 6, but with a larger opening in the substrate.

In Fig. 1, 10 shows a device for electrical contact animals of cells 11 which are in suspension in a reservoir 12 in a Vorratsbe liquid 14 held in suspension.

The reservoir 12 is connected to a changeover valve 15 through which the liquid 14 is passed into a flow channel 16 , in which the electrical contacting of the cells 11 takes place in a manner to be described.

An inlet 17 for a washing solution is also connected to the switching valve 15 , so that the flow channel 16 can be flushed with the washing solution after measurement on a cell 11 .

A sensor unit 18 is initially arranged along the flow channel 16 , via which the parameters of the cells 11 are measured. The sensor unit 18 is followed by a microperfusion unit 19 , via which perfusion solutions 20 can be introduced into the flow channel 16 in order to perfuse a contacted cell, that is to say to wash it with test substances.

The micro perfusion unit 19 is followed by a positioning unit 21 , via which cells 11 can be positioned in a contacting and measuring unit 22 and immobilized there.

The positioning unit 21 is, for. B. controlled as a function of output signals from the sensor unit 18 .

In Fig. 1, a conventional patch clamp tip 23 is indicated in the contacting and measuring unit 22 , via the measurement solutions on the outside of a membrane of the cell 11 and in tracellular measurements can be carried out automatically in a manner yet to be described.

Before the patch clamp tip 23 , a further sensor unit 18 'can be arranged, which is arranged in the exemplary embodiment shown in FIG. 1 in the contacting and measuring unit 22 . The further sensor unit 18 'can locate individual cells in the flow channel 16 via optical and other methods and control the suctioning of this single cell with its measuring channel, as will be described in detail further below.

While the sensor unit 18 serves to select a cell to be sucked in, it is the task of the further sensor unit 18 'to locate a single cell and to determine its current location in such a way that it comes into contact with the patch via the positioning unit 21 and / or by suction. Clamp syringe 23 can be brought ge.

The contacting and measuring unit 22 is followed by a second sensor unit 24 and a control 25 for a transport flow of the liquid 14 in the flow channel 16 indicated at 26. The flow channel 16 finally opens into a collecting container 27 for liquid 14 and washing solution 17 .

Finally, in Fig. 1 a sequence control and Meßda tenverarbeitung 28 is shown, via which the individual components of the device are controlled and functionally connected.

Via the sequence control and measurement data processing 28 , the transport flow 26 is set so that a single cell flows past the sensor unit 18 , measured there with respect to certain parameters and then positioned in the contacting and measuring unit in the event of their selection by the positioning unit 21 becomes.

The positioning unit 21 can, for. B. be a pair of laser tweezers, or build up an electromagnetic field via which antibodies coupled to magnetic particles that sit on the surface of the cells 11 are transported to the measuring and discharge point.

In addition, it is also possible instead of or in addition to the positioning unit 21 in the contacting and measuring unit 22 to provide a hydrodynamic negative pressure for immobilizing a cell 11 , as will be described below in connection with FIG. 2.

The sensor unit 18 is designed to detect an optical, electrical, geometric or other biological, chemical or physical cell parameter and to report it to the sequence control and measurement data processing 28 , which thereupon the positioning unit 21 and / or the contacting and measuring unit 22 controls accordingly if the cell identified as suitable on the basis of its parameter has reached the area of the contacting and measuring unit 22 due to the known transport flow 26 .

The contacting and measuring unit 22 is designed in Mikrosystemtech technology as a microstructured part, so it has very small dimensions.

In Fig. 2 is a section of the device 10 of FIG. 1 in an enlarged scale and in a schematic side view shows ge. The flow channel 16 has a substrate 31 as a wall, in which an opening 32 is provided, on which a cell 11 to be contacted is positioned. An opening 33 opens into the opening 32 , which is connected to a pump 34 , via which a hydrodynamic negative pressure can be set in the opening 32 for drawing in a cell 11 .

Around the opening 32 , facing the cell 11, a functional coating 35 is arranged on the substrate 31 , which in addition to the suction channel 33 serves to immobilize a cell 11 . When the cell 11 has been positioned on the opening 32 , the vacuum of the pump 34 is switched off, so that the cell 11 only adheres to the functional coating 35 when it responds to the coating, otherwise it is washed away by the transport flow 26 . For the actual measurement, however, the pump 34 is then switched on again, so that the cell 11 is securely immobilized on the opening 32 .

The first positioning on the opening 32 thus served only to test whether the cell 11 responds to the functional coating 35 which ensures chemical immobilization or immobilization by means of antibodies immobilized on its part. The functional coating 35 can also be a magnetic coating which holds antibodies coupled to magnetic particles, which in turn sit on the cell 11 . This first positioning of the cell 11 on the opening 32 can take place not only via the intake duct 33 but also via the positioning unit 21 from FIG. 1.

Remote from the cell 11 sits on the substrate 31 from a removable contact unit 36 on which an interchangeable contact tip 37 is arranged. The contact tip 37 bears with its upper end 38 against a cell membrane 39 of the cell 11 and bulges it slightly inwards.

The contact tip 37 is penetrated by a contact channel 41 , which opens at the upper end 38 of the contact tip 37 . At the other end, the contact channel 41 is connected to a pump 42 , by means of which a hydrodynamic vacuum can be set.

In the contact channel 41 protrudes an electrode 43 which is in the contact channel 41 existing in the electrically conductive liquid speed with the cell membrane 39 in electrical connection. A reference electrode 44 , which projects into the flow channel 16 in the region of the opening 32, serves as the counter electrode.

The cell 11 is immobilized on the opening 32 through the suction channel 33 , while the contacting - as in the usual patch-clamp technique - takes place by applying a slight negative pressure in the contact channel 41 . If a pressure surge is generated in the contact channel 41, the membrane spot circumscribed by the contact tip 37 is perforated, so that intracellular measurements and stimulations are possible.

A perfusion channel 45 also opens into the flow channel 16 , through which a perfusion solution 20 can be passed to the immobilized and contacted cell 11 , in order to flush it with test substances and to measure the reaction of the cell 11 to the test substances.

In Fig. 3, the process of contacting and measuring / stimulating a cell 11 is shown in a schematic image sequence.

In the first picture in the upper row, the flow channel 16 is flowed through by a liquid 14 which carries a cell 11 with it. Liquid 14 is also flushed into the interior of the flow channel 16 through the suction channel 33 .

When the cell 11 has reached the area of the opening 32 , a hydrodynamic negative pressure is generated via the intake duct 33 , so that the cell 11 is drawn towards the opening 32 , as shown in the second image of the first row.

The last picture of the first row shows that the cell 11, with its membrane 39 arched inwards, lies on the upper end 38 of the contact tip 37 , as is also the case with the classic patch-clamp technique. In contrast to classic technology, contacting in the new device and the new method takes place in that the cell 11 is transported to the contact tip and is automatically immobilized there.

In the second line in the first picture it can be seen that a negative pressure is applied to the contact channel 41 , so that the cell mobilized in the cell is contacted in the manner customary in patch-clamp technology. If the negative pressure in the contact channel 41 is increased in a pulse-like manner, the membrane spot opens up, so that an intracellular measurement is possible, as shown in the second image of the second line.

When the measurement / stimulation has ended, the negative pressure in the contact channel 41 is switched off and an excess pressure is applied in the suction channel 43 , so that the cell 11 is released again into the transport flow 26 and transported away by this.

In the third line in the left picture it can be seen that the contact tip is then removed from the opening 32 and a new contact tip 37 is inserted, which is indicated in the second picture of the third line.

After the contact tip 37 has been replaced, the flow channel 16 and the suction channel 33 and the contact channel 41 are first flushed with liquid in order to prepare the system for contacting a new cell 11 .

The exchange of the contact tip 37 is carried out automatically by means of a micromanipulator, so that with the new device 10 many cells can be contacted and measured / stimulated one after the other in an automated manner.

In Fig. 4, a first, very simply constructed execution example for an interchangeable contact tip 37 is shown.

In the exemplary embodiment shown, the contact tip is a conventional patch clamp tip 23 , as is already known from FIG. 1. The patch clamp tip 23 is cast into a plastic block 46 , which is pressed from the outside against the substrate 31 ge, so that the patch clamp tip 23 protrudes into the opening 32 . The opening 32 is closed to the outside in a liquid-tight manner by means of sealing means 47 , which are preferably formed by Syl gard ^TM .

The patch clamp tip 23 protrudes somewhat in the exemplary embodiment shown, out over the substrate 31 into the flow channel 16 , so that a cell 11 sucked via the suction channel 33 onto the opening 32 forms a bulge indicated at 48 in its cell membrane 39 , such as it is also set by hand using the patch clamp technique.

In FIG. 4, in the upper installation of the plastic block is shown in the flow channel 16 46, while shown in the lower view of the plastic block 46 which is arranged in the contact channel 41 electrode 43. A micromanipulator, which is only shown schematically, acts on the plastic block 46 . The micromanipulator 49 presses the plastic block 45 against the substrate 31 and holds the plastic block 46 under pressure in position with the substrate 31 so that the liquid-tight seal already mentioned is achieved.

The contact unit 36 shown in Fig. 4 is very simple to build, it consists essentially of a conventional patch clamp tip 23 , which was poured into the plastic block 46 sen.

Another embodiment of a contact unit 36 is shown in FIG. 5. The contact unit 36 from FIG. 5 has an interchangeable upper part 51 and a reusable lower part 52 . The contact tip 37 with the contact channel 41 is arranged on the upper part 51 , a chamber 53 being provided in the lower part 52 , into which the electrode 43 projects. A first section 54 of the contact channel 41 also leads into the chamber 53 , the second, funnel-shaped section 55 of which is arranged in the upper part 51 .

Between the upper part 51 and the lower part 52 are the sealing means 47 known from FIG. 4, which are also arranged between the upper part 51 and the substrate 31 .

The contact unit 36 of Fig. 5 is very simple and easy to position, only the upper part 51 has to be replaced to replace the contact tip 37 .

For this purpose, the entire contact unit 36 is removed from the substrate 31 via a micromanipulator, whereupon the upper part 51 is replaced and the lower part 52 is pressed against the substrate 31 again with a new upper part 51 . This exchange can take place automatically, which brings a noticeable time saving compared to manual exchange.

In the exemplary embodiment of the contact unit 36 from FIG. 6, an annular space 58 is formed between the upper part 51 and the substrate 31 , which surrounds the contact tip 37 . The annular space 58 leads through an opening 59 in the upper part 51 to a first section 61 of the intake duct 33 , which is also arranged in the contact unit 36 in this embodiment, for example.

When the upper part 51 has been pressed against the substrate 31 , for which the lower part 52 is pressed against the upper part 51 , the already known sealing means 47 again ensure a liquid-tight seal.

In the embodiment of FIG. 6, all hydro dynamic connections are provided on the lower part 52 , the suction channel 33 opening in the opening in a known manner.

In the exemplary embodiment for the contact unit 36 in FIG. 7, compared to the exemplary embodiment in FIG. 6, the annular channel 58 to the very thin substrate 31 is closed off by a membrane 62 which forms a passage 63 around the contact tip 37 .

In comparison to FIG. 6 it can be seen that the opening 32 in the substrate 31 is considerably larger, so that the contact unit 36 can be positioned very easily on the substrate 31 . Otherwise, the contact unit 36 from FIG. 7 corresponds to the contact unit 36 from FIG. 6.

Claims (37)

1. Device for electrically contacting biological cells ( 11 ) in suspension in a liquid ( 14 ), with a substrate ( 31 ) having at least one opening ( 32 ), with means ( 35 , 33 ) for immobilizing a cell ( 11) on the opening (32), preferably one in the orifice (32) opens the intake passage (33) through which at the opening (32) is a hydrodynamic low pressure can be generated for immobilizing a cell (11) on the opening (32) and at least one electrode ( 43 ) for the electrical contacting of an immobilized cell ( 11 ), characterized in that a contact unit ( 36 ) with a contact tip ( 37 ) is arranged at the opening ( 32 ), the upper end ( 38 ) of which protrudes into the opening ( 32 ) that it comes into contact with a cell membrane ( 39 ) of an immobilized cell ( 11 ), the contact tip ( 37 ) has a contact channel ( 31 ) opening at its upper end ( 38 ), Via which a hydrodynamic negative pressure can be exerted on the cell membrane ( 39 ), and the electrical de ( 43 ) is electrically connected to the contact channel ( 41 ). 2. Device according to claim 1, characterized in that the contact unit ( 36 ) is arranged removably at the opening ( 32 ). 3. Apparatus according to claim 1 or 2, characterized in that the contact tip ( 23 , 37 ) is replaceably angeord net. 4. Device according to one of claims 1 to 3, characterized in that the suction channel ( 33 ) in the substrate ( 31 ). 5. Device according to one of claims 1 to 3, characterized in that the suction channel ( 33 ) extends at least in sections in the contact unit ( 36 ). 6. Device according to one of claims 1 to 5, characterized in that the contact unit has an interchangeable, the contact tip ( 37 ) having upper part ( 51 ) and a reusable, the electrode ( 43 ) and one of the most he section ( 54 ) of the contact channel ( 41 ) having lower part ( 52 ). 7. Device according to one of claims 1 to 6, characterized in that the contact unit ( 36 ) in the region of the opening ( 32 ) bears on the outside of the substrate ( 31 ) and the opening ( 32 ) closes liquid-tight to the outside. 8. Device according to claims 6 and 7, characterized in that the upper part ( 51 ) bears under pressure on the outside of the substrate ( 31 ) and the lower part ( 52 ) under pressure on the upper part ( 51 ). 9. The device according to claim 8, characterized in that between the substrate ( 31 ) and upper part ( 51 ) and / or between the upper part ( 51 ) and lower part ( 52 ), a sealant ( 47 ) is seen before. 10. The device according to claim 9, characterized in that the sealant ( 47 ) is selected from the group: Silicon paste, two-component elastomer, polydimethylsiloxane elastomer. 11. Device according to one of claims 6 to 10, characterized in that in the lower part ( 52 ) a chamber ( 53 ) is provided, into which the electrode ( 43 ) protrudes and a first section ( 54 ) of the contact channel ( 41 ) empties. 12. The apparatus according to claim 11, characterized in that in the upper part ( 51 ) associated with the chamber ( 53 ), the contact tip ( 37 ) penetrating, funnel-shaped two-th section ( 55 ) of the contact channel ( 41 ) is arranged. 13. Device according to one of claims 6 to 12, characterized in that between the upper part ( 51 ) and the substrate ( 31 ) around the contact tip ( 37 ) around an annular space ( 58 ) is formed, which points to the lower part ( 52 ) has an opening ( 59 ) which is assigned to a first section ( 61 ) of the intake duct ( 33 ) in the lower part ( 51 ). 14. The apparatus according to claim 13, characterized in that the annular space ( 58 ) on the substrate ( 31 ) facing with egg ner membrane ( 62 ) is sealed, in which the contact tip ( 37 ) surrounds the passage ( 63 ) at a distance hen is. 15. The apparatus according to claim 14, characterized in that the membrane ( 62 ) on the upper part ( 51 ) is attached. 16. The device according to one of claims 1 to 15, characterized in that a reference electrode ( 44 ) projects into the liquid ( 14 ) in the region of the opening ( 32 ). 17. Device according to one of claims 1 to 16, characterized in that on the substrate ( 31 ) around the opening ( 32 ) around a functional coating ( 35 ) is provided as a means for immobilizing the cells ( 11 ). 18. The apparatus according to claim 17, characterized in that the functional coating is selected from the group pe: polyanions; polycations; polyethyleneimine; antibody against cell surface molecules such as integrins (e.g. Fibro nectin), or lectins; and magnetic coating. 19. Device according to one of claims 1 to 18, characterized by a positioning unit ( 21 ) to position a cell ( 11 ) over the opening ( 32 ). 20. The apparatus according to claim 19, characterized in that the positioning unit ( 21 ) comprises laser tweezers. 21. The apparatus according to claim 19, characterized in that the positioning unit ( 21 ) builds up an electromagnetic field. 22. The apparatus according to claim 19, characterized in that the positioning unit ( 21 ) generates a hydrodynamic force. 23. Device according to one of claims 1 to 22, characterized in that the substrate ( 31 ) is an integral part of a wall of a flow channel ( 16 ) through which the liquid ( 14 ) with a defined and / or adjustable transport flow ( 26 ) is directed to the opening ( 32 ). 24. Device according to one of claims 1 to 23, characterized by a sensor unit ( 18 ) which responds to a parameter of the cells ( 11 ) and controls a positioning unit ( 21 ) and / or the suction channel ( 33 ). 25. The device according to claim 24, characterized in that the parameter is selected from the group: optical Pa parameters such as fluorescence, absorption, reflect xion, diffraction, refraction; geometric parameters as for for example size, length, diameter, shape; electrical Parameters such as capacitance, resistance; and biological, chemical, physical cell parameters. 26. The apparatus of claim 24 or 25, characterized in that the sensor unit ( 18 ) upstream of the opening ( 32 ) is arranged. 27. The apparatus according to claim 23, characterized in that a micro perfusion unit ( 19 ) is arranged upstream of the opening ( 32 ) in order to wash around an immobilized cell ( 11 ) with test substances. 28. Device according to one of claims 1 to 27, characterized ge indicates that they are at least partially in microsystem technology is made. 29. A method for electrically contacting biological cells ( 11 ) in suspension in a liquid ( 14 ), comprising the steps:

• - Immobilizing a cell ( 11 ) above an opening ( 32 ) provided in a substrate ( 31 ), preferably by generating a hydrodynamic vacuum, and
• - Contacting the immobilized cell ( 11 ) via at least one electrode ( 43 ),

characterized in that for contacting the immobilized cell ( 11 ) through a contact tip ( 37 ) projecting into the opening ( 32 ) onto a cell membrane ( 39 ) of the cells ( 11 ), a hydrodynamic negative pressure is exerted, the electrode ( 43 ) through the contact tip ( 37 ) through electrically with the cell ( 11 ). 30. The method according to claim 29, characterized in that the contact tip ( 37 , 23 ) is exchanged before each contact. 31. The method according to claim 29 and 30, characterized in that the liquid ( 14 ) with adjustable Transportströ tion ( 26 ) through a flow channel ( 16 ) to the opening ( 32 ) is passed. 32. The method according to any one of claims 29 to 31, characterized in that the cell ( 11 ) is immobilized by adhering to a functional coating ( 35 ) which is arranged around the opening ( 32 ) on the substrate ( 31 ). 33. The method according to any one of claims 29 to 32, characterized in that the cell ( 11 ) is positioned by a positioning unit ( 21 ) over the opening ( 32 ). 34. A method according to claim 33, characterized in that prior to positioning a cell (11) measured a parameter of the cell (11) and the cell (11) is selected for positioning in dependence upon the parameters. 35. The method according to claim 32 and 33, characterized in that a cell ( 11 ) is selected in that it is positioned by means of the positioning unit ( 21 ) on the functional coating ( 35 ) and then released by the positioning unit ( 21 ) is, so that only cells remain immobilized that respond to the functional coating ( 35 ). 36. The method according to claim 35, characterized in that the cell is immobilized in addition to the functional coating ( 35 ) by hydrodynamic vacuum. 37. The method according to any one of claims 29 to 36, characterized in that the cell is removed from the opening ( 32 ) by the underpressure applied for immobilization being built up to an overpressure.